DESCRIPTION (applicant's abstract): The PI's laboratory has focused on the study of novel lipid mediators and their role in cell regulation. Recent work in the PI's laboratory has led to the identification and doning of two yeast sphingosine-1-phosphate phosphatases (SPPase). These enzymes dephosphorylate and inactivate an extremely important bioactive lipid mediator namely sphingosine-1-phosphate (S1P). These enzymes also regulate the cellular levels of two other bioactive sphingolipids, sphingosine (SPH) and ceramide. S1P has recently emerged as a critical lipid second messenger and ligand for the Edg receptor family whereby it mediates cell growth and inhibits apoptosis. SPH is also a potent lipid mediator that inhibits cell growth and inhibits protein kinase C. Ceramide has also recently emerged as a key sphingolipid mediator involved in the regulation of mammalian and yeast stress responses. Regulation of cellular S1P, SPH, and ceramide levels therefore becomes critical to regulation of cell growth. Our specific hypothesis is that SPPase is a key regulator of SiP, SPH, and ceramide levels and of cellular responses to these bioactive lipids. Therefore, the main goals of this proposal are focused on: 1) doning the murine and human SPPases and determining their tissue, cellular and subcellular distribution. We have already identified an EST and begun the mammalian cloning, we will tag these enzymes, purify them, prove their activity, and study their tissue and cellular distribution. 2) Characterizing and studying the regulation of these novel mammalian SPPases, by determining KM for substrate, pH profile, cation dependence, and substrate specificity. At the cellular level we will study its regulation in response to inducers of cell growth and apoptosis. 3) determining the role of SPPase in regulaling cellular levels of SiP, SPH, and ceramide by determining changes in these bioactive lipids in cells where the SPPase has been overexpressed or knocked-out; and 4) determining the role of SPPase in cell regulation, by determining the effect of over-expression or knock-out of SPPase on cell growth and apoptosis. These studies should provide fundamental insight into the biochemical action of this enzyme. These studies will also allow important insight into mechanisms of its regulation and its role in the regulation of basal sphingolipid metabolism. Finally, these studies should provide key insight into the physiologic function of this enzyme and its role in cell regulation.